The present invention relates to the field of molecular biology, and in particular to the detection of nucleic acids and cells. Methods and compositions for detection of extremely low amounts of nucleic acids and cellular materials with ATP based detection systems are described.
Methods for producing large amounts of recombinant protein are well known. As the recombinant protein industry has developed, the need for various quality control assays has arisen. An example is the need for the quantitation of nucleic acids present in recombinant protein preparations. Current guidelines require that the amount of nucleic acid present in recombinant therapeutic proteins be less than 10 pg of DNA per daily dose of recombinant protein. Therefore, methods for detecting extremely low amounts of nucleic acids are needed. Such methods would also find widespread use for the quantitation of DNA in forensic samples.
Several methods of detecting low levels of nucleic acid have been described. The first method is based on classical hybridization techniques. This method utilizes radio-labeled nucleic acid probes which bind to the DNA of interest. However, this method has several disadvantages including poor reproducibility, generation of large amounts of waste reagent, and high background levels caused by nonspecific binding. Furthermore, this technique is generally inappropriate for determining the presence of low amounts of DNA of unknown sequence.
A second method of detecting nucleic acid utilizes fluorescent dyes capable of intercalating into nucleic acids. However, many interfering substances such as detergents, proteins, and lipids affect the reproducibility of the signal generated by this method.
A third method of detecting low levels of DNA utilizes biotinylated single-stranded DNA binding protein (SSB), streptavidin, an anti-DNA antibody fused to urease, and biotinylated nitrocellulose as reagents. This assay is commercially available from Molecular Devices and described in Kung et al., Picogram Quantitation of Total DNA Using SNA-Binding Proteins in a Silicon Sensor-Based System, Anal. Biochem. 187: 220-27 (1990). The assay is performed by incubating the streptavidin, biotin-SSB, and the anti-DNA antibody together, allowing a complex to be formed. The complex is then captured on the biotinylated filter, washed, and the amount of captured urease is read. This method is highly sensitive but has several disadvantages. These disadvantages include costly reagents and the need for extensive controls.
A fourth method involves the depolymerization or degradation of nucleic acids and detection of ATP by luciferase. Polynucleotide polymerases are responsible for the synthesis of nucleic acids in cells. These enzymes are also capable of catalyzing other reactions as described in Deutscher and Kornberg, Enzymatic Synthesis of Deoxyribonucleic Acid, J. Biol. Chem. 244(11):3019-28 (1969). Many, but not all, polymerases are able to depolymerize nucleic acid in the presence of either phosphate or pyrophosphate.
U.S. Pat. No. 4,735,897 describes a method of detecting polyadenylated messenger RNA (poly(A)-mRNA). Depolymerization of poly(A)-mRNA in the presence of phosphate has been shown to result in the formation of ADP, which can be converted by pyruvate kinase or creatine phosphokinase into ATP. RNA may also be digested by a ribonuclease to AMP, converted to ADP by adenylate kinase, and then converted to ATP by pyruvate kinase.
The ATP so produced is detected by a luciferase detection system. In the presence of ATP and O2, luciferase catalyzes the oxidation of luciferin, producing light which can then be quantitated using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin.
The presence of ATP-generating enzymes in all organisms also allows the use of a luciferase system for detecting the presence or amounts of contaminating cells in a sample, as described in U.S. Pat. No. 5,648,232. For example, ADP may be added to a sample suspected of containing contaminating cells. The ADP is converted by enzymes of the cell into ATP which is detected by a luciferase assay, as described above. The disadvantage of this method is the relative instability of the ADP substrate.
What is needed in the art are reliable, cost-effective methods of detecting extremely low levels of nucleic acids, cells, and cellular material in a wide variety of samples. The present invention discloses novel methods for detecting low quantities of DNA, RNA and of cells. These methods take advantage of novel combinations of pyrophosphorolysis or enzymatic degradation of nucleic acids, conversion of dNTPs to ATP, the conversion of AMP directly to ATP, amplification of ATP to increase sensitivity, depolymerization of oligonucleotide probes, and optimized reaction conditions.
A need exists for quality control assays for proteins produced by recombinant methods. Current guidelines suggest that preparations of recombinant protein should contain less than 10 picograms of nucleic acid. There is also a need to be able to quantitate extremely low levels of nucleic acids in forensics samples. Therefore, it is an object of the present invention to provide methods for detecting low amounts of nucleic acids and low numbers of cells or cellular material. It is also an object of the invention to provide compositions for the detection of nucleic acids and kits for the detection of nucleic acids.
In one embodiment of the present invention a method is provided for detecting and/or assaying deoxyribonucleic acid in a reaction containing phosphate, adenosine 5xe2x80x2-diphosphate, or a combination thereof. The method comprises depolymerizing the nucleic acid at a terminal nucleotide by enzymatically cleaving the terminal internucleoside phosphodiester bond and reforming same with a pyrophosphate molecule to form a deoxyribonucleoside triphosphate molecule according to the following reaction:
NAn+PPixe2x86x92NAnxe2x88x921+dNTP
catalyzed by a DNA polymerase or reverse transcriptase selected from the group consisting of T4 polymerase, Taq polymerase, AMV reverse transcriptase, and MMLV reverse transcriptase. In a quantitative assay for nucleic acids, the depolymerizing step is repeated essentially to completion or equilibrium to obtain at least two nucleoside triphosphate molecules from a strand of minimally three nucleotides. For detection of DNA, the depolymerizing step need not be repeated if there are sufficient nucleic acid molecules present to generate a signal. The next step involves enzymatically transferring terminal 5xe2x80x2 phosphate groups from the deoxyribonucleoside triphosphate molecules to an adenosine 5xe2x80x2-diphosphate molecule to form adenosine 5xe2x80x2-triphosphate according to the following reaction:
dNTP*+ADPxe2x86x92NDP+ATP*
catalyzed by nucleoside diphosphate kinase and wherein P* is the terminal 5xe2x80x2 phosphate so transferred. The final step is the detection of the ATP, either by a luciferase detection system or NADH detection system. The depolymerizing step and phosphate transferring step may optionally be performed in a single pot reaction. If greater sensitivity is desired, the ATP molecules produced by the phosphate transferring step or the NTPs produced by the depolymerizing step may be amplified to form a plurality of ATP molecules.
In another embodiment of the present invention, a method is provided for detecting polyadenylated mRNA in a reaction containing pyrophosphate. The polyadenylated mRNA is first depolymerized at a terminal nucleotide by enzymatically cleaving the terminal internucleoside phosphodiester bond and reforming same with a pyrophosphate molecule to form a free ATP molecule according to the following reaction:
NAn+PPixe2x86x92NAnxe2x88x921+ATP
catalyzed by poly(A) polymerase. In a quantitative assay for RNA, the depolymerizing step is repeated essentially to completion or equilibrium to obtain at least two nucleoside triphosphate molecules from a strand of minimally three nucleotides. For detection of DNA, the depolymerizing step need not be repeated if there are sufficient nucleic acid molecules present to generate a signal. The ATP molecules so formed are then detected with either a luciferase detection system or a NADH detection system. The sensitivity of the reaction may be increased by optionally amplifying the ATP molecules.
In another embodiment of the present invention, a method is provided for selectively detecting and/or assaying poly(A) mRNA in a reaction containing pyrophosphate, adenosine 5xe2x80x2-diphosphate, or a combination thereof. In this method, a complementary oligo (dT) probe is hybridized to poly(A) mRNA to form an RNA-DNA hybrid. The oligo (dT) strand of the RNA-DNA hybrid is then depolymerized at the terminal nucleotide by enzymatically cleaving the terminal internucleotide phosphodiester bond and reforming same with a pyrophosphate molecule to form deoxythymidine 5xe2x80x2-triphosphate. According to the following reaction:
xe2x80x83TTn+PPixe2x86x92TTnxe2x88x921+dTTP
catalyzed by a reverse transcriptase. In a quantitative assay for nucleic acids, the depolymerizing step is repeated essentially to completion or equilibrium to obtain at least two nucleotide triphosphate molecules from a strand of minimally three nucleotides. For detection of DNA, the depolymerizing step need not be repeated if there are sufficient nucleic acid molecules present to generate a signal. Next, the phosphate groups from the deoxythymidine 5xe2x80x2-triphosphate are enzymatically transferred to adenosine 5xe2x80x2-diphosphate molecules to form ATP molecules according to the following reaction:
dTTP*+ADPxe2x86x92dTDP+ATP*
catalyzed by NDPK, wherein P* is the terminal 5xe2x80x2 phosphate so transferred. Finally, the ATP so formed is detected by a luciferase detection system or an NADH detection system. If increased sensitivity is desired, the terminal phosphate of the dTTP may be transferred to ADP to form ATP as above followed by an amplification of the resulting ATP.
In another embodiment of the present invention, a method is provided of detecting DNA in a reaction containing phosphoribosylpyrophosphate, adenosine 5xe2x80x2-diphosphate, or a combination thereof. In this method, free deoxyribonucleoside monophosphate molecules are produced from the nucleic acid by digestion with a nuclease. A pyrophosphate group is then enzymatically transferred from phosphoribosylpyrophosphate molecules to the deoxyadenosine monophosphate molecules to form deoxyadenosine triphosphate molecules according to the following reaction:
dAMP+PRPPxe2x86x92dATP+ribose-5-PO4
catalyzed by phosphoribosylpyrophosphate synthetase. Next, the terminal 5xe2x80x2 phosphate groups from the deoxyadenosine triphosphate molecules are enzymatically transferred to adenosine 5xe2x80x2-diphosphate molecules to form ATP molecules according to the following reaction:
xe2x80x83dATP*+ADPxe2x86x92dADP+ATP*
catalyzed by NDPK wherein P* is a terminal 5xe2x80x2 phosphate so transferred. The ATP so produced may be detected by a luciferase detection system or an NADH detection system. If desired, the pyrophosphate transferring step and the phosphate transferring step may be performed in a single pot reaction. If increased sensitivity is required, the ATP molecules may be amplified.
Another embodiment of the present invention provides a method of detecting RNA in a reaction containing phosphoribosylpyrophosphate. Free ribonucleoside monophosphate molecules are produced by digestion of RNA with a nuclease. Next, a pyrophosphate molecule from phosphoribosylpyrophosphate molecules is enzymatically transferred to the adenosine monophosphate molecules to form adenosine triphosphate molecules according to the following reaction:
NMP+PRPPxe2x86x92NTP+ribose-5-PO4
catalyzed by phosphoribosylpyrophosphate synthetase. The ATP so produced is then detected by a luciferase detection system or an NADH detection system. If increased sensitivity is required, the ATP so produced may be amplified.
Another embodiment of the present invention provides a method for determining the presence and/or amount of cells and cellular material present in the sample. In this method, the contents of cells are released to form a cell lysate. Phosphate donor molecules and adenosine 5xe2x80x2-monophosphate molecules are then added to the cell lysate so that adenosine 5xe2x80x2-diphosphate molecules are produced by the enzymatic transfer of an phosphate group from the donor to the adenosine 5xe2x80x2-monophosphate according to the following reaction:
Dxe2x88x92P+AMPxe2x86x92D+ADP
catalyzed by endogenous enzymes present in the cell lysate. ATP is then produced by the enzymatic transfer of a phosphate from the donor molecules to adenosine 5xe2x80x2-diphosphate molecules according to the following reaction:
Dxe2x88x92P+ADPxe2x86x92D+ATP
also catalyzed by endogenous enzymes present in the cell lysate sample. The adenosine 5xe2x80x2-triphosphate so produced is then detected by either a luciferase detection system or an NADH detection system. The phosphate donor of this embodiment may be either deoxycytidine 5xe2x80x2-triphosphate, deoxyguanidine 5xe2x80x2-triphosphate, or deoxythymidine 5xe2x80x2-triphosphate.
The present invention further provides a composition of matter for producing adenosine 5xe2x80x2-triphosphate from DNA, pyrophosphate, and adenosine 5xe2x80x2-diphosphate. This composition comprises a mixture of nucleoside diphosphate kinase and a nucleic acid polymerase which are provided in a concentration sufficient to catalyze the production of ATP from DNA at about picogram to microgram amounts of DNA.
The present invention, also provides a composition of matter for producing adenosine triphosphate from DNA, phosphoribosylpyrophosphate, and adenosine 5xe2x80x2-diphosphate. This composition comprises a mixture of a phosphoribosylpyrophosphate synthetase and nucleoside diphosphate kinase in a sufficient concentration to catalyze the production of adenosine triphosphate from about picogram to microgram amounts of DNA.
The present invention provides various kits for nucleic acid detection. First, a kit is provided which contains reagents for the detection of DNA by pyrophosphorolysis. The kit contains a vessel containing a nucleic acid polymerase and a vessel containing a nucleoside disphosphate kinase. The nucleic acid polymerase and nucleoside diphosphate kinase may be provided in the same container. Second, a kit is provided which contains reagents for the detection of nucleic acid by nuclease digestion. The kit contains a vessel containing phosphoribosylpyrophosphate synthetase and a vessel containing a nuclease. Third, a kit is provided which contains reagents for the detection of RNA by pyrophosphorolysis. The kit contains a vessel containing poly(A)-polymerase. Fourth, a kit containing reagents for the detection of DNA by nuclease digestion is provided. This kit contains a vessel containing phosphoribosylpyrophosphate synthetase and a vessel containing nucleoside disphosphate kinase. The phosphoribosylpyrophosphate synthetase and nucleoside diphosphate kinase may optionally be provided in the same container.
An embodiment of the present invention further provides a kit containing reagents for the detection of cells and/or cellular material in a sample. The kit contains a vessel containing adenosine 5xe2x80x2-monophosphate and a vessel containing a high energy phosphate donor which may not be utilized by luciferase.
The present invention also provides a method of amplifying a nucleoside triphosphate molecule in a reaction containing adenosine 5xe2x80x2-monophosphate molecules, high energy phosphate donor molecules, or a combination thereof. In this method, the terminal 5xe2x80x2 phosphate group from a nucleoside triphosphate molecule (XTP) present in the sample is enzymatically transferred to an adenosine 5xe2x80x2-monophosphate molecule added to the sample to form adenosine 5xe2x80x2-diphosphate molecules and nucleoside diphosphate molecules (XTP, either a ribonucleoside or deoxyribonucleoside triphosphate) according to the following reaction:
XTP+AMPxe2x86x92XDP+ADP
catalyzed by a first enzyme which may be either nucleoside monophosphate kinase or adenylate kinase. Next, a phosphate from a high energy phosphate donor molecule which may not be utilized by the first enzyme is enzymatically transferred to the adenosine 5xe2x80x2-diphosphate molecules to form adenosine 5xe2x80x2-triphosphate molecules according to the following reaction:
xe2x80x83ADP+Dxe2x88x92Pxe2x86x92ATP+D
catalyzed by nucleoside diphosphate kinase or pyruvate kinase. These two steps are then repeated until the desired level of amplification is achieved. The high energy phosphate donors may be either dCTP or AMP-CPP for NDPK and PEP for pyruvate kinase.
The present invention also provides a method for detecting deoxyribonucleic or ribonucleic acid in a reaction containing pyrophosphate, adenosine 5xe2x80x2-monophosphate, and a high energy phosphate donor, or a combination thereof, in a single pot reaction. First, nucleic acid is depolymerized at a terminal nucleotide by enzymatically cleaving the terminal internucleotide phosphodiester bond with a pyrophosphate molecule to form a free ribonucleoside (XTP) or deoxynucleoside triphosphate molecule (XTP) according to reaction 1:
NA+PPixe2x86x92NAnxe2x88x921+XTP
catalyzed by a polymerase. The depolymerizing step is repeated to obtain at least two nucleoside triphosphate molecules. The ribonucleoside triphosphate molecules or deoxyribonucleoside triphosphate molecules are then amplified by enzymatically transferring the terminal 5xe2x80x2 phosphate group from the nucleoside triphosphate molecule formed in reaction 1 to an adenosine 5xe2x80x2-monophosphate to produce an adenosine 5xe2x80x2-diphosphate molecule and a nucleoside 5xe2x80x2-diphosphate molecule (XDP) according to reaction 2 catalyzed by a first enzyme:
XTP+AMPxe2x86x92XDP+ADP.
Next, a phosphate group from a high energy phosphate donor molecule, which is not a substrate for the first enzyme, is enzymatically transferred to the adenosine 5xe2x80x2-diphosphate molecules produced in a reaction to produce adenosine 5xe2x80x2-triphosphate molecules according to reaction 3 catalyzed by a second enzyme:
ADP+Dxe2x88x92Pxe2x86x92ATP+D.
The two amplification steps are repeated until the desired level of amplification is achieved. Enzyme 1 in this method may be either adenylate kinase or nucleoside monophosphate kinase, while enzyme 2 may be either pyruvate kinase or nuceloside diphosphate kinase.
The present invention provides a method for detecting extremely low levels of various nucleic acids including both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in biological samples, especially samples of recombinant proteins. The extreme sensitivity, reproducibility, ease of conducting the reactions, and speed of conducting the reactions represent major advantages over methods currently in use for low level detection of nucleic acids.
The detection method may be divided into three general steps. The first step is the production of the following nucleosides: nucleoside monophosphates (XMPs) including the ribonucleoside monophosphates (NMPs) adenosine 5xe2x80x2-monophosphate (AMP), guanosine 5xe2x80x2-monophosphate (GMP), uridine 5xe2x80x2-monophosphate (UMP), and cytidine 5xe2x80x2-monophosphate (CMP); deoxyribonucleoside monophosphates (dNMPs) including deoxyadenosine 5xe2x80x2-monophosphate (dAMP), deoxyguanosine 5xe2x80x2-monophosphate (dGMP), deoxythymidine 5xe2x80x2-monophosphate (dTMP), and deoxycytidine 5xe2x80x2-monophosphate (dCMP); nucleoside triphosphates (XTPs) including the ribonucleoside triphosphates (NTPs) adenosine 5xe2x80x2-triphosphate (ATP), guanosine 5xe2x80x2-triphosphate (GTP), uridine 5xe2x80x2-triphosphate (UTP), and cytidine 5xe2x80x2-triphosphate (CTP); and the deoxyribonucleoside triphosphates (dNTPs) including deoxyadenosine 5xe2x80x2-triphosphate (dATP), deoxyguanosine 5xe2x80x2-triphosphate (dGTP), deoxythymidine 5xe2x80x2-triphosphate (dTTP), and cytidine 5xe2x80x2-triphosphate (dCTP). The NMPs and dNMPs are produced by nuclease digestion and the NTPs and dNTPs by depolymerization by pyrophosphorolysis. The second step, used when the initial substrate is DNA, is the transfer of the terminal phosphate from the dNTPs to ADP to form ATP. The optional step of XTP amplification may be performed at this stage to increase the sensitivity of the detection system especially when measuring samples containing low levels of DNA in the range of 1-10 picograms of nucleic acid. The third step is detection of ATP by a suitable detection method. Examples of such detection systems are the luciferase detection system and NADH-based detection system.
Nucleic acid polymerases generally catalyze the elongation of nucleic acid chains. The reaction is driven by the cleavage of a pyrophosphate released as each nucleoside is added. Each nucleoside triphosphate has three phosphate groups linked to carbon 5 of ribose or deoxyribose. The addition of a nucleoside to a growing nucleic acid results in formation of a internucleoside phosphodiester bond. This bond is characterized in having a 3xe2x80x2 linkage to carbon 3 of ribose or deoxyribose and a 5xe2x80x2 linkage to carbon 5 of ribose or deoxyribose. Each nucleoside is added through formation of a new 3xe2x80x2 linkage, so the nucleic acid strand grows in a 5xe2x80x2 to 3xe2x80x2 direction. The 5xe2x80x2 end of the nucleic acid is characterized by a phosphate group attached to carbon 5.
Several polymerases are also known to catalyze the reverse of the polymerization process. This reverse reaction is called pyrophosphorolysis. The pyrophosphorolysis activity of DNA polymerase was demonstrated by Deutscher and Kornberg, Enzymatic Synthesis of Deoxyribonucleic Acids, J. Biol. Chem. 244: 3019-28 (1969). Other nucleic acid polymerases capable of pyrophosphorolysis include DNA polymerase a, DNA polymerase xcex2, T4 DNA polymerase, Taq polymerase, Klenow fragment, AMV reverse transcriptase, and MMLV reverse transcriptase. However, not all polymerases are known to possess pyrophosphorolysis activity. For example, poly(A) polymerase has been reported to not catalyze pyrophosphorylation. (See Sippel, Eur. J. Biochem. 37:31-40 (1973).)
A mechanism of pyrophosphorolysis has been suggested for RNA polymerase. It is believed that the partial transfer of a Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleoside phosphodiester bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester as described in Rozovskaya et al. Biochem. J. 224: 645-50 (1984). The internucleoside phosphodiester bond is enzymatically cleaved by the addition of pyrophosphate to the nucleoside 5xe2x80x2 phosphate and a new phosphodiester bond is formed between the pyrophosphate and the nucleoside monophosphate.
The pyrophosphorolysis reaction can be summarized as follows:
NAn+PPixe2x86x92NAnxe2x88x921+XTPxe2x80x83xe2x80x83Reaction 1
wherein NA is a nucleic acid, PPi is pyrophosphate and XTP is either a deoxyribonucleoside triphosphate molecule or a ribonucleoside triphosphate molecule. The reaction may then be repeated to obtain at least two XTP molecules. It should be noted that the reaction may be repeated on the same nucleic acid molecule or on a plurality of different nucleic acid molecules.
Preferred reaction mixes for depolymerization by pyrophosphorolysis, including suitable buffers for each nucleic acid polymerase analyzed are disclosed in the Examples. Under these conditions, sufficient NTP or dNTP is produced to accurately detect or assay extremely low amounts of nucleic acids (5-15 picograms.)
Even though the preferred reaction conditions for polymerization and depolymerization by pyrophosphorolysis are similar, the rates of these reactions vary greatly. For example, AMV and RLV reverse transcriptases catalyze pyrophosphorolysis under optimal conditions at a rate of about fifty- to one hundred-fold less than polymerization as demonstrated in Srivastavan and Modak, J. Biol. Chem. 255(5): 2000-04 (1980). Thus, the high eficiency of the pyrophosphorolysis reaction was unexpected and appears to be asscociated with extremely low levels of DNA substrate in contrast to previous DNA pyrophosphorolysis studies conducted on much higher amounts of DNA.
The pyrophosphorolysis activity of different nucleic acid polymerases also varies. T4 polymerase appears to possess the greatest pyrophosphorolysis activity as measured by a luciferase assay for ATP produced by pyrophosphorolysis. Pyrophosphorolysis using T4 polymerase resulted in about a 10 fold increase in light production as compared to MMLV-RT and a 4 fold increase in light production as compared to Taq polymerase.
The detection of nucleic acids at low picogram levels is generally enhanced by fragmenting or partially digesting the nucleic acid. Preferably, fragmentation is accomplished by sonication or restriction enzyme digestion of the nucleic acid, forming a plurality of smaller nucleic acid fragments. This step probably enhances detection because the pyrophosphorolysis reaction only proceeds from the DNA ends, as demonstrated in the Examples. Providing a greater number of DNA ends means that more reactions are occurring at any one time. It should be noted that DNA ends may be present within a molecule as well as at the end of a linear DNA fragment. For example, polymerases may catalyze pyrophosphorolysis from a gap in a DNA segment or a nick in a DNA segment. The type of enzyme used for pyrophosphorolysis and the type of substrate determine whether fragmentation is necessary. For instance, the data set forth in the Examples demonstrate that fragmenting greatly increases detection of plasmid DNA when Tag polymerase is used, but does not effect detection when T4 polymerase is used. However, when chromosomal DNA is the substrate, fragmentation increases detection from both enzymes.
The type of cut made by restriction enzyme digestion also affects the pyrophosphorolysis activity of different nucleic acid polymerases. For example, MMLV-RT and Tag polymerase can catalyze pyrophosphorolysis of DNA fragments with 5xe2x80x2 overhangs, but not 3xe2x80x2 overhangs. In contrast, T4 DNA polymerase catalyzes both 3xe2x80x2 and 5xe2x80x2 end overhang and blunt end mediated pyrophosphorolysis. T4 polymerase is thus a preferred enzyme for pyrophosphorolysis. When other nucleic acid polymerases are utilized for pyrophosphorolysis of restriction enzyme treated DNA, care must be taken to match the overhang specificity of the polymerase with the type of overhang created by the restriction endonuclease.
It must be noted that sequence specificity of pyrophosphorolysis for single stranded DNA has been previously noted during sequencing by Tabor and Richardson, J. Biol. Chem. 265 (14): 8322-28 (1990). The sequence specificity of the pyrophosphorolysis reaction was noted when some dideoxynucleoside terminated sequence fragments were shown to be more susceptible to degradation by phosphorolysis than other fragments.
Further, the type of polymerase used in the pyrophosphorolysis reaction must be matched to the correct nucleic acid substrate. In general, DNA polymerases and reverse transcriptases are preferred for depolymerizing DNA, and RNA polymerases are preferred for depolymerizing RNA. Reverse transcriptases are preferred for depolymerizing RNA-DNA hybrids.
Applicants have further demonstrated that poly(A) polymerase may catalyze pyrophosphorolysis, even though no such reaction has been previously reported. In fact, poly(A) polymerase has been widely reported to not catalyze pyrophosphorolysis. See, for example, Sippel, Eur. J. Biochem. 37:31-40 (1973) and Sano and Feix, Eur J. Biochem. 71:577-83 (1976). Surprisingly, the applicants show that under the proper reaction conditions poly(A) polymerase catalyzes phosphorolysis. Preferably, the manganese chloride present in the previously reported buffers is omitted, the concentration of sodium chloride is decreased, and the pH is lowered from about 8.0 to about 7.5. Most preferably, poly(A) polymerase pyrophosphorolysis reaction buffer contains about 50 mM Tris-Cl pH 7.5, 10 nmM MgCl2, 50 mM NaCl, and 2 mM NaPPi (sodium pyrophosphate).
It is important to note that the depolymerization reaction is the reverse of the polymerization reaction. Therefore, as increasing amounts of free nucleoside triphosphates are produced by depolymerization, a state of equilibrium may theoretically be attained in which polymerization and depolymerization reactions are balanced. Alternatively, where small amounts of nucleic acid are detected, the reaction may go essentially to completion without reaching equilibrium, i.e. the nucleic acid depolymerized into its constituent subunits nucleotides by greater than 90%. This is important in a quantitative test because the total amount of nucleotides released is proprtional to the amount of signal generated in the detection assay. When used for qualitative detection of nucleic acid, it is not necessary that the reaction reach equilibrium or go essentially to completion provided a threshold level of nucleotides are produced. The mixture of nucleoside triphosphate molecules produced by depolymerization will preferably be converted to adenosine triphosphate as described below. For either detection or assay, a detectable threshold level of 6xc3x97107 adenosine triphosphate molecules must be provided for detection of light from a typical luciferase assay.
In a preferred embodiment of the present invention for detecting nucleic acids, nucleic acid polymerase and PPi are added to a sample containing less than 1 xcexcg nucleic acid, down to less than about 10 pg of nucleic acid. To increase the sensitivity of the DNA detection, the DNA may be fragmented by treatment with a restriction endonuclease or by sonication. Next, the nucleic acid is degraded by pyrophosphorolysis releasing free NTPs or dNTPs. Enzymes useful in the pyrophosphorolysis reaction include AMV reverse transcriptase, MMLV reverse transcriptase, DNA polymerase alpha and beta, Taq polymerase, T4 DNA polymerase, Klenow fragment and poly(A) polymerase. Most preferably, T4 polymerase is utilized for DNA pyrophosphorolysis reactions because of its recognition of 3xe2x80x2 and 5xe2x80x2 overhangs and blunt ends and high processivity as noted above.
Luciferase, which is part of the preferred ATP detection system, is inhibited by pyrophosphate (PPi). Therefore, care must be taken not to transfer a highly inhibiting amount of PPi to the ATP detection reaction. Preferably, the amount PPi carried over to the ATP detection reaction results in a concentration of PPi in the luciferase detection reaction of less than about 100 xcexcM, although less than about 10 xcexcM is desirable. Therefore, the amount of PPi utilized in the pyrophosphorolysis reaction will be determined by the size of the aliquot which is taken for use in the luciferase detection system. The aliquot size may vary, but the amount of PPi transferred or carried over to the luciferase detection reaction should correspond to the PPi concentration parameters described above so that the concentration of PPi is at least below about 100 xcexcM, and preferably below about 10 xcexcM.
In another embodiment, the nucleic acids may be first degraded into NMP or dNMP by exonuclease digestion according to the following reaction:
NAn+H2Oxe2x86x92NAnxe2x88x921+XMPxe2x80x83xe2x80x83Reaction 2
wherein NA is a nucleic acid, XMP is either a deoxyribonucleoside monophosphate or ribonucleoside monophosphate, and n is the number of nucleosides in the nucleic acid.
Nuclease digestion may be accomplished by a variety of nucleases including S1 nuclease, nuclease BAL 31, mung bean nuclease, exonuclease III and ribonuclease H. Nuclease digestion conditions and buffers may be found in the Product Literature available from commercial sources, or as disclosed in the Examples.
After digestion with the nuclease, the NMPs or dNMPs are converted to NTPs or dNTPs respectively. U.S. Pat. No. 4,375,897 describes the detection of RNA by digestion with nucleases followed by conversion to NTP. This method utilizes a two-step scheme in which adenylate kinase converts AMP to ADP, and pyruvate kinase then converts ADP to ATP. This method is essentially limited to the detection of poly(A) MRNA because no mechanism is suggested for conversion of dNTPs to ATP, the preferred substrate for luciferase. Nuclease digestion or phosphorolysis of DNA results in a mixture of dNTPs which do not act as efficient substrates for luciferase.
In the biosynthesis of purine and pyrimidine mononucleosides, phosphoribosylpyrophosphate (PRPP) is the obligatory ribose-5xe2x80x2-phosphate donor. PRPP itself is formed in a reaction catalyzed by PRPP synthetase through the transfer of pyrophosphate from ATP to ribose-5-phosphate. This reaction is known to be reversible as described in Sabina et al., Science 223: 1193-95 (1984).
In the present invention, the NMP or dNMP produced by nuclease digestion is preferably converted directly to NTP or dNTP by the enzyme PRPP synthetase in the following reactions:
XMP+PRPPxe2x86x92XTP+ribose-5-PO4xe2x80x83xe2x80x83Reaction 3
wherein XMP is either adenosine monophosphate or deoxyadenosine monophosphate and XTP is either a adenosine triphosphate or deoxyadenosine triphosphate. Preferably, this reaction produces a detectable threshold level of at least 6xc3x97107 adenosine triphosphate molecules.
In this reaction, the pyrophosphate group of PRPP is enzymatically transferred to XMP molecules, forming XTP molecules. Reaction conditions and buffers are set forth in the Examples. When RNA is the substrate, the ATP produced may be directly detected.
Utilization of the PRPP reaction in the nucleic acid detection system has several advantages over the prior art. First, only one step is necessary to convert an AMP or dAMP to a ATP or DATP, which simplifies the detection system. Second, contamination of the detection reaction with exogenous ATP, ADP, or AMP is less likely.
The dNTPs produced by pyrophosphorolysis or nuclease digestion followed by pyrophosphorylation can theoretically be used directly as substrates for luciferase, allowing detection of the nucleic acid. However, the preferred substrate for luciferase is ATP as demonstrated by Moyer and Henderson, Nucleoside Triphosphate Specificity of Firefly Luciferase, Anal. Biochem. 131:187-89 (1983). When DNA is the initial substrate, a nucleoside diphosphate kinase (NDPK) is conveniently utilized to catalyze the conversion of dNTPs to ATP by the following general reaction:
dNTP*+ADPxe2x86x92dNDP+ATP*xe2x80x83xe2x80x83Reaction 4
wherein dNTP is a mixture of deoxyribonucleoside triphosphates and dNTP is the corresponding deoxyribonucleoside triphosphate. In the reaction, the terminal 5xe2x80x2-triphosphate (P*) of the dNTP is transferred to ADP to form ATP.
Enzymes catalyzing this reaction are generally known as nucleoside diphosphate kinases (NDPK). NDPKs are ubiquitous, relatively nonspecific enzymes. For a review of NDPK, see Parks and Agarwal, in The Enzymes, Volume 8, P. Boyer Ed. (1973). The conversion of NTPs or dNTPs to ATP by NDPK is preferably accomplished by adding NDPK and a molar excess of ADP over the amounts of NTPs or dNTPs expected to be produced by pyrophosphorolysis, or nuclease digestion followed by is pyrophosphorylation by PRPP synthetase. Alternatively, if an amplification scheme is used, a molar excess of AMP may be used as the preferred substrate. The utilization of ADP requires optimization of the amount of ADP added. Too much ADP results in high background levels. A reaction containing NDPK contains about 0.01 to 0.50 xcexcM ADP, preferably about 0.05 xcexcM ADP. Illustrative buffers and reaction components are set forth in the Examples.
As an optional step, the NTP, dNTP, or ATP generated by the pyrophosphorolysis or nuclease digestion schemes may be amplified to give even greater sensitivity. Amplification may be required when utilizing detection systems other than luciferase or when increased levels of signal are needed for detection by a less sensitive luminometer. Amplification of NTP means a continuous reaction wherein 1 NTP gives rise to 2 NTPs, which can be cycled to yield 4 NTPs and so on. When AMP is added to feed the amplification reaction, ATP will accumulate while the amount of original NTP remains the same. PCT publication WO 94/25619 and Chittock et al., Anal. Biochem., 255:120-6 (1998), incorporated herein by reference, disclose amplification systems for ATP characterized by the following coupled reactions:
C1+S1xe2x86x922C2 and 2C2+2xe2x86x922C1
2C1+2S1xe2x86x924C2 and 4C2+4S2+E2xe2x86x924C1
4C1+4S1xe2x86x928C2 and 8C2+8S2+E2xe2x86x928C1xe2x80x83xe2x80x83Reaction 5
wherein C1 is the target compound present in a sample to be amplified, S1 is the amplification substrate, E1 is a catalytic enzyme capable of utilizing C1 and S1 to produce C2, S2 is an energy donating substrate, and E2 is a catalytic enzyme capable of utilizing C2 and S2 to produce C1, which then recycles through the reaction. According to this reaction scheme, each pass through the coupled reaction doubles the amount of C1, which can be subsequently detected. Patent Application GB 2,055,200 discloses an amplification system utilizing adenylate kinase and pyruvate kinase.
In designing a coupled ATP amplification reaction for use in nucleic acid detection, two main requirements must be considered. First, E1 must not be able to utilize the high energy phosphate donor utilized by E2. If E1 can utilize the high energy phosphate donor, the ATP amplification reaction would proceed in the absence of NTP or dNTP produced as a result of pyrophosphorolysis or nuclease digestion followed by pyrophosphorylation. This would result in the undesirable occurrence of false positive results. Second, a molar excess of the added high energy phosphate donor must be provided as compared to the amount of dXTP or XTP expected in the reaction. Third, E1 must be able to utilize either the NTP, dNTP, or ATP produced in step 1 by pyrophosphorolysis or nuclease digestion of the nucleic acid.
The amplification system of the present invention may be characterized, as follows:
xe2x80x83XTP+AMPxe2x86x92XDP+ADP
ADP+Dxe2x88x92Pxe2x86x92ATPxe2x80x83xe2x80x83Reaction 6
wherein Dxe2x88x92P is a high energy phosphate donor and E1 and E2 are enzymes capable of catalyzing the transfer of phosphates from an XTP to AMP and from the Dxe2x88x92P to ADP, respectively. The ATP so produced may reenter the reaction (as XTP) and the reaction repeated until the substrates are exhausted or equilibrium is reached, resulting in the production of two ATPs for every ATP supplied to or generated by the reaction. Note that when the target XTP is any nucleoside triphosphate other than ATP the initial pass through the cycle yields only 1 ATP which then reenters the cycle to produce two ATP, which reenter the cycle to produce 4 ATP and so on. Preferably, the amplification reaction produces a detectable threshold level of 6xc3x9710 7 adenosine triphosphate molecules.
The XTP in reaction 6 is a ribonucleoside triphosphate or deoxyribonucleoside triphosphate, which may preferably be ATP provided by pyrophosphorolysis (Reaction 1) or created from XTP by NDPK conversion of ADP to ATP (Reaction 5) or provided by nuclease digestion coupled with pyrophosphorylation (Reaction 4) followed by NDPK conversion to ATP (Reaction 5). It must be appreciated, however, that when an amplification step is utilized for a DNA substrate, the step of converting dNTP to ATP is inherent in the amplification system. Therefore, a separate converting step is not needed.
A nucleoside monophosphate kinase (NMPK) or adenylate kinase is preferably utilized as enzyme 1 (E1). NMPKs occur as a family, each of which is responsible for catalyzing the phosphorylation of a particular NMP. Until recently, it was generally thought that ATP and DATP were preferred phosphate donors. However, Shimofuruya and Suzuki Biochem. Intl. 26(5):853-61 (1992) recently demonstrated that at least some NMPKs can utilize other phosphate donors such as CTP and UTP. Enzyme 2 (E2) is preferably nucleoside diphosphate kinase (NDPK) or pyruvate kinase. NDPK""s generally catalyze the transfer of the terminal 5xe2x80x2-triphosphate of NTPs to NDPs to form NTPs. Pyruvate kinase catalyzes the transfer of phosphate from phosphoenolpyruvate to ADP to form ATP. These enzymatic activities are utilized in the amplification reaction to transfer a phosphate group from a high energy phosphate donor (Dxe2x88x92P) to either ADP or an NDP.
A high energy phosphate donor (Dxe2x88x92P) that may be used by E2 but not by E1 is required. When E2 is NDPK, dCTP or xcex1, xcex2 methylene adenosine 5xe2x80x2-triphosphate (AMP-CPP) may be utilized as Dxe2x88x92P. When E2 is pyruvate kinase, phosphoenol pyruvate (PEP) is the preferred high energy phosphate donor. The ability of NDPK to utilize these substrates at efficiencies allowing production of minute quantities of ATP was not known. It is surprising that these high-energy phosphate donors utilized with NMPK and adenylate kinase meet the requirements of the amplification reaction when the recent literature suggests that NMPK (E1) may utilize phosphate donors other than ATP or dATP. The nonspecificity of adenylate kinase is also well known. The high energy phosphate donor and/or AMP must be provided in a molar excess as compared to the amount of ATP or dNTP expected present in the sample so that the high energy phosphate donor is not recycled at an appreciable rate. Additional buffers and reaction components are included in the Examples.
The third step of nucleic acid detection is detection of the NTP, dNTP or amplified ATP. Two well known detection systems include: the light emitting luciferase detection system, and the NADH light adsorption detection system (NADH detection system).
The ATP so produced is detected by a luciferase detection system. In the presence of ATP and O2, luciferase catalyzes the oxidation of luciferin, producing light which can then be quantitated using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin. The light can be detected by a luminometer.
The preferred ATP detection buffer, which will be referred to as LAR buffer, is formulated by mixing 19.1 ml of deionized water; 800 xcexcl of 0.5M Tricine, pH 8.0; 70 xcexcl of 1M MgSO4; 4 xcexcl of 0.5M EDTA; 0.108g of DTT (dithiothreitol); 0.003 g of Luciferin; and adjusting the pH to 7.8 if necessary. Preferably, about 5 to 10 nanograms of recombinant luciferase (Promega Lot 6414002) is used in the reaction. Greater amounts of luciferase have a tendency to increase non-specific background. Applicants also have shown that deleting coenzyme A from the LAR reaction mix decreases background.
In the NADH detection system, a combination of two enzymes, phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase, are used to catalyze the formation of NAD from NADH in the presence of ATP. ATP is measured as a loss in fluorescence intensity because NADH is fluorescent while NAD is not. Examples of NADH based ATP assays are disclosed in U.S. Pat. Nos. 4,735,897, 4,595,655, 4,446,231 and 4,743,561, and UK Patent Application GB 2,055,200, all incorporated herein by reference.
Certain of the above reactions may be performed as single pot reactions. A single pot reaction is a reaction wherein at least two enzymes (E1 and E2) with catalytic activity are present in the same reaction mix and act on one or more substrates) (S1 and S2). The reactions catalyzed by the enzymes may occur simultaneously where E1 acts on S1 and E2 acts on S2 successfully. Alternatively, the reactions catalyzed by E1 and E2 may occur in a step-wise or coupled manner where E1 acts on S1 to produce an intermediate S2i and E2 then acts on S2i. Of course, such a coupled reaction may also be essentially simultaneous.
The ability to utilize combinations or mixtures of the enzymes of the present invention in single pot reactions is surprising in light of the extremely low levels of nucleic acid detection which are achieved. This low level detection is possible even though some enzymes are used under less than optimal conditions. As previously described, it was necessary to optimize the concentration of PPi utilized in the pyrophosphorolysis reactions so that luciferase would not be inhibited. Therefore, aliquots from the NMP, dNMP, NTP, dNTP and ATP producing reactions may be directly added to LAR buffer for luciferase detection without any purification of the reaction products. The luciferase reaction is not poisoned or otherwise quenched by the components of the reactions. This desirable feature allows high throughput screening with a minimal amount of time and effort, and also allows great flexibility in the design of the overall detection schemes.
Preferably, the pyrophosphorolysis reaction producing dNTP and the NDPK catalyzed reaction in which the NTPs or dNTPs are converted to ATP may be performed in a single pot reaction in the nucleic acid polymerase buffer. NDPK activity is sufficient to convert dNTP to ATP even though the polymerase buffer conditions are suboptimal for NDPK activity. The polymerase enzyme and NDPK may both be present initially in the reaction, or the NDPK may be added directly to the reaction after an incubation period sufficient for the production of NTP or dNTP. A nucleic acid polymerase and NDPK may be provided in the same vessel or mixture for use in the reactions described above. The mixture preferably contains the nucleic acid polymerase and NDPK in a concentration sufficient to catalyze the production of ATP when in the presence of a nucleic acid, pyrophosphate and ADP. Preferably, the polymerase is provided in a concentration of about 1 to 100 units/xcexcl, most preferably at about 5 units/xcexcl. Preferably, the NDPK is provided in a concentration of 0.1 to 100 units/xcexcl, most preferably at about 5 units/xcexcl. Preferably, the mixture is greater than 99% pure.
Similarly, the PRPP synthetase and NDPK reactions can be performed in a single pot reaction in the PRPP synthetase buffer. Again, NDPK activity is sufficient even though conditions for NDPK activity are suboptimal. The nuclease digested sample containing free NMPs and dNMPs may be added to a reaction mix initially containing PRPP synthetase and NDPK, or added to a PRPP synthetase reaction followed by addition to a reaction mix containing NDPK and the luciferase detection reaction components. The preferred buffers and reaction components may be found in the Examples. PRPP synthetase and NDPK may be provided in the same vessel or mixture for use in the reactions described above. The mixture preferably contains the PRPP synthetase and NDPK in a concentration sufficient to catalyze the production of ATP when in the presence of phosphoribosylpyrophosphate and ADP. Preferably, the NDPK is provided in a concentration of 0.1 to 100 units/xcexcl, most preferably at about 5 units/xcexcl. Preferably, the PRPP synthetase is provided in a concentration of 0.001 to 10 units/xcexcl, most preferably at about 0.01 units/xcexcl If amplification is desired, the PRPP synthetase reaction must be heat inactivated, otherwise the PRPP synthetase would convert the added AMP to ATP. Preferably the mixture is greater than 99% pure.
The pyrophosphorolysis reaction and amplification reaction may also be performed in a single pot reaction. In this single pot reaction, the polymerases may be AMV reverse transcriptase, MMLV reverse transcriptase, DNA polymerase alpha or beta, Taq polymerase, T4 DNA polymerase, Klenow fragment or poly(a) polymerase, a first enzyme for converting AMP to ADP may be myokinase (adenylate kinase) or NMPK, and a second enzyme for converting ADP to ATP may be pyruvate kinase or NDPK. The reaction must be fed AMP, preferably Apyrase treated AMP so that background due to contaminating ADP and ATP is minimized. Preferably 1xcexcl of 1U/xcexcl Apyrase may be added to 19 xcexcl of 10 mM AMP, followed by incubation at room temperature for 30 minutes and heat inactivation of the Apyrase by incubation at 70xc2x0 C. for 10 minutes. High energy phosphate donors must also be added to the reaction. When pyruvate kinase is utilized phosphoenolpyruvate is added, while when NDPK is utilized dCTP is added. Preferably, the high energy phosphate donor is added about 15 minutes after a preincubation with the polymerase, although this is not necessary. These reactions may characterized as follows:
NAn+PPixe2x86x92NAnxe2x88x921+XTP
XTP+AMPxe2x86x92ADP+XDP
ADP+Dxe2x88x92Pxe2x86x92ATP+Dxe2x80x83xe2x80x83Reaction 7
wherein NA is a nucleic acid, XTP is a nucleoside triphosphate, either a deoxynucleoside or ribonucleoside triphosphate, XDP is a nucleoside diphosphate, either a deoxynucleoside or ribonucleoside diphosphate, and Dxe2x88x92P is a high energy phosphate donor. It should be appreciated that this reaction produces ATP, the preferred substrate for luciferase, from dNTPs. The amplification reaction proceeds as described in reaction 7 to produce a detectable threshold level of 6xc3x97107 adenosine triphosphate molecules. Preferably, the polymerase is provided in a concentration of about 1 to 100 units/xcexcl, most preferably at about 5 units/xcexcl. Preferably, the NDPK is provided in a concentration of 0.1 to 100 units/xcexcl, most preferably at about 1 unit/xcexcl. Preferably, the Preferably, the mixture is greater than 99% pure.
In another embodiment, the reactions described above may be used to selectively detect poly(A) mRNA according to the following scheme. First oligo(dT) primers are hybridized to the poly(A) tails of the mRNA to form a DNA-RNA hybrid. Next, a pyrophosphorolysis reaction is performed using reverse transcriptase (RT). Reverse transcriptases which may be used in the present invention include Mouse Mammary Leukemia Virus (MMLV) RT, Avian Myeloma Virus (AMV) RT and Rous Sarcoma Virus (RSV) RT. An advantage of this detection system is that these RTs catalyze pyrophosphorolysis of double stranded nucleic acid and double stranded RNA-DNA hybrids, but not single stranded nucleic acids. Thus, the amount of poly(A) RNA in a total cellular RNA sample be determined. The pyrophosphorolysis reaction produces dTTP according to the following reaction:
TTn+PPixe2x86x92TTnxe2x88x921+dTTP;xe2x80x83xe2x80x83Reaction 8
wherein TTn is oligo(dT) and PPi is pyrophosphate.
The dTTP can be converted to ATP by NDPK as described in reaction 4 above, optionally amplified, and detected as described above.
In another embodiment, the reactions described above may be used to detect the presence of cells in a sample. U.S. Pat. No. 5,648,232, incorporated herein by reference, describes a method of detecting cells in a sample. That method takes advantage of adenylate kinase activity, which is present in all living organisms. Briefly, a sample suspected of containing microorganisms or other living cells is subjected to conditions causing cell lysis. ADP is then added to the lysate, which is converted by endogenous adenylate kinase activity to ATP by the following reaction:
ADP+ADPxe2x86x92ATP+AMPxe2x80x83xe2x80x83Reaction 9
The ATP produced by this reaction is then detected by the luciferase assay system.
The present invention also provides a method of detecting the presence of cells in a lysate of a sample suspected of containing cellular material by using different substrates. This system takes advantage of a coupled reaction catalyzed by endogenous adenylate kinase activity (AK) and NDPK activity according to the following reaction scheme:
AMP+Dxe2x88x92Pxe2x86x92D+ADP and
ADP+Dxe2x88x92Pxe2x86x92ATP+D.xe2x80x83xe2x80x83Reaction 10
wherein Dxe2x88x92P is a high energy phosphate donor added to the cell lysate and AMP is adenosine monophosphate added to the cell lysate sample. In this reaction, adenosine 5xe2x80x2-diphosphate molecules are produced by the enzymatic transfer of a phosphate group from the high energy phosphate donor molecules (Dxe2x88x92P) to the added adenosine 5xe2x80x2-monophosphate (AMP) molecules. Then, adenosine 5xe2x80x2-triphosphate is produced by the enzymatic transfer of phosphate from Dxe2x88x92P molecules to the adenosine 5xe2x80x2-diphosphate molecules according to the following general reaction catalyzed by endogenous enzymes present in the cell lysate sample
Co-optimization of the concentrations of nucleosides added to the samples was necessary to optimize light output from these reactions. About 1 mM to 80 mM AMP and 1 mM to 100 mM dCTP may be added to the test sample, and preferably about 10 mM AMP and 100 mM dCTP may be added to the test sample. After addition of nucleosides to the sample, the samples are preferably incubated at room temperature for about 10 to 60 minutes, and light output from the samples determined by a luminometer. Other preferred buffers and reactions components may be found in the Examples.
This system has an important advantage over previously described cell detections systems. The AMP and dCTP are much more stable than ADP, so the results are more reproducible.
In another aspect of the present invention, a nucleic acid detection test kit is provided for performing the pyrophosphorolysis nucleic acid detection method. The nucleic acid detection test kit comprises the essential reagents required for the method of the nucleic acid detection invention. For nucleic acid detection by pyrophosphorolysis, the kit includes a vessel containing an enzyme capable of catalyzing pyrophosphorolysis such as Taq polymerase, T4 polymerase, AMV reverse transcriptase, MMLV reverse transcriptase, or poly(A) polymerase. The concentration of polymerase is 0.1 to 100 units/xcexcl, preferably about 5 units/xcexcl. Kits for use in DNA detection also include a vessel containing nucleoside diphosphokinase and a vessel containing ADP. Preferably, these reagents are free of contaminating ATP and adenylate kinase. The NDPK is provided in concentration of about 0.1 to 100 units/xcexcl, preferably about 1.0 units/xcexcl. The contaminants may be removed by dialysis or Apyrase treatment. Optionally, the kit may contain vessels with reagents for amplification of dNTPs or NTP to ATP. Amplification reagents include pyruvate kinase, adenylate kinase, NMPK, NDPK, AMP as the amplification substrate, and dCTP or AMP-CPP as high-energy phosphate donors. The kit may be packaged in a single enclosure including instructions for performing the assay methods. The reagents are provided in containers and are of a strength suitable for direct use or use after dilution. A standard set may also be provided to allow quantitation of results. Test buffers for optimal enzyme activity may be included. Most preferably, the NDPK and nucleic acid polymerase are provided in the same reaction mix so that a single pot reaction may be performed consistently.
In another aspect of the present invention, a nucleic acid detection kit is provided for performing the nuclease digestion nucleic acid detection method of the present invention. This test kit comprises the essential reagents required for this method. These reagents include a nuclease, PRPP synthetase, PRPP, NDPK, and ADP together with luciferase and luciferin. The nuclease is provided in a concentration of about 1 to 500 units/xcexcl, preferably about 20 units/xcexcl. The PRPP synthetase is provided in concentration of about 0.01 units/xcexcl to 10 units/xcexcl, preferably about 0.1 units/xcexcl. Preferably, the kit includes all these reagents with luciferase and luciferin being provided as a single reagent solution. Most preferably, the PRPP synthetase and NDPK are provided in a single reaction mix so that a single pot reaction containing these two enzymes may be performed, simplifying the detection method. The kit is in the form of a single package preferably including instructions to perform the method of the invention. The reagents are provided in vessels and are of a strength suitable for direct use or use after dilution. Preferably, buffers which support the optimal enzyme activity are provided. Optionally, reagents for amplification of the ATP signal may be provided as described in the previous kit.
In another aspect of the present invention, a test kit is provided for determining the presence of microorganisms or other cells in a test sample. This test kit comprises the essential reagents required for the method. These reagents include a high-energy phosphate donor which may not be utilized by luciferase, preferably dCTP, and AMP together with luciferase and luciferin. Preferably, the kit includes all these reagents with luciferase and luciferin being provided in the same solution. Preferably, the reagents are free of contaminating components such as adenylate kinase and ATP that would cause a false positive test. A cell lysis cocktail may be provided for efficiently releasing the contents of the target cells for each of the assays intended. For prokaryotic microorganisms, only a cationic detergent is needed. For fungal spores or eukaryotic cells assays, a further nonionic detergent reagent is included. Reagents are provided in vessels and are of a strength suitable for direct use or use after dilution. A buffer solution for diluting the cell samples may also be provided.